WAF1 induced by isopropyl-1-thio-␤-D-galactopyranoside were similar to those induced by NGF. Therefore, we have identified a signal transduction pathway that is activated by NGF; proceeds through NOS, p53, and p21 WAF1 to block cell proliferation; and is required for neuronal differentiation by PC12 cells.

Targeting of gene regulatory factors to specific intranuclear sites may be critical for the accurate control of gene expression. The acute myelogenous leukemia 8;21 (AML1͞ETO) fusion protein is encoded by a rearranged gene created by the ETO chromosomal translocation. This protein lacks the nuclear matrix-targeting signal that directs the AML1 protein to appropriate gene regulatory sites within the nucleus. Here we report that substitution of the chromosome 8-derived ETO protein for the multifunctional C terminus of AML1 precludes targeting of the factor to AML1 subnuclear domains. Instead, the AML1͞ETO fusion protein is redirected by the ETO component to alternate nuclear matrix-associated foci. Our results link the ETO chromosomal translocation in AML with modifications in the intranuclear trafficking of the key hematopoietic regulatory factor, AML1. We conclude that misrouting of gene regulatory factors as a consequence of chromosomal translocations is an important characteristic of acute leukemias. M odifications in nuclear morphology are a hallmark of tumor cells and are used for diagnosis. However, there is limited knowledge of the subnuclear changes that accompany or contribute to tumor-related alterations of nuclear architecture. Biochemical evidence indicates that the protein composition of the nuclear matrix is modified in tumor cells (1-7). The nuclear matrix is a scaffold that provides a means for localizing genes and regulatory factors throughout the nuclear space (8-11). Consequently, analysis of targeting signals that direct regulatory factors to nuclear matrix-associated subnuclear sites (12) may provide insight into nuclear structure-function relationships that are compromised in cancer.The acute myelogenous leukemia (AML) transcription factor AML1 1 is a key regulator of hematopoiesis (13,14). Numerous cytogenetic abnormalities that involve genes encoding AML1 or its partner core binding factor ␤ have been identified in AML and acute lymphocytic leukemia (15-18). The frequent 8;21 translocation produces a chimeric protein (AML1͞ETO) in which the C terminus of AML1 is replaced by the unrelated ETO (MTG8) protein (19-23). The 8;21 translocation occurs in approximately 15% of AML in adult patients (24-26).The normal form of the AML1 protein has an N-terminal region containing a runt homology DNA-binding domain (27) and a multifunctional C-terminal region that supports transactivation, repression, and intranuclear targeting (28-34). Alternative splicing of the AML1 gene can generate several protein isoforms. The predominant isoform AML1B (480 aa) contains a 31-aa nuclear matrix-targeting signal (NMTS) in the C terminus. This targeting signal is necessary and sufficient to direct the factor to nuclear matrix-associated subnuclear sites that support transcription (30). Many of these AML1 sites are localized together with the hyperphosphorylated active form of RNA polymerase II o , and this association requires the presence of the runt homology domain of AML1B (35).The AML1͞ETO fusion product is expre...

The runt-related transcription factors (RUNX/Cbfa/AML) are essential for cellular differentiation and fetal development. C-terminal truncations of RUNX factors that eliminate the targeting of these factors to subnuclear foci result in lethal hematopoietic and skeletal phenotypes. Here we demonstrate that in living cells the RUNX C-terminus is necessary for the dynamic association of RUNX into stable subnuclear domains. Time-lapse fluorescence microscopy shows that RUNX1 and RUNX2 localize to punctate foci that remain stationary in the nuclear space. By fluorescence recovery after photobleaching assays, both proteins are shown to dynamically associate at these subnuclear foci, with a 10 second half-time of recovery. A truncation of RUNX2, removing its intranuclear targeting signal (NMTS), increases its mobility by an order of magnitude, resulting in a half-time of recovery equivalent to that of EGFP alone. We propose that the dynamic shuttling of RUNX factors in living cells to positionally stabilized foci, which is dependent on the C-terminus, is a component of the mechanism for gene regulation in vivo.

The architecturally associated subnuclear organization of nucleic acids and cognate regulatory factors suggest functional interrelationships between nuclear structure and gene expression. Mechanisms that contribute to the spatial distribution of transcription factors within the three-dimensional context of nuclear architecture control the sorting of regulatory information as well as the assembly and activities of sites within the nucleus that support gene expression. Vitamin D control of gene expression serves as a paradigm for experimentally addressing mechanisms that govern the intranuclear targeting of regulatory factors to nuclear domains where transcription of developmental and tissue-specific genes occur. We will present an overview of molecular, cellular, genetic, and biochemical approaches that provide insight into the trafficking of regulatory factors that mediate vitamin D control of gene expression to transcriptionally active subnuclear sites. Examples will be presented that suggest modifications in the intranuclear targeting of transcription factors abrogate competency for vitamin D control of skeletal gene expression during development and fidelity of gene expression in tumor cells.

No abstract